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Background Brucellosis, as a serious zoonotic infectious disease, has been recognized as a re-emerging disease in the developing countries worldwide. In china, the incidence of brucellosis is increasing each year, seriously threatening the health of humans as well as animal populations. Despite a quite number of diagnostic methods currently being used for brucellosis, innovative technologies are still needed for its rapid and accurate diagnosis, especially in area where traditional diagnostic is unavailable. Methodology/Principal findings In this study, a total of 22 B cell linear epitopes were predicted from five Brucella outer membrane proteins (OMPs) using an immunoinformatic approach. These epitopes were then chemically synthesized, and with the method of indirect ELISA (iELISA), each of them displayed a certain degree of capability in identifying human brucellosis positive sera. Subsequently, a fusion protein consisting of the 22 predicted epitopes was prokaryotically expressed and used as diagnostic antigen in a newly established brucellosis testing method, nano-ZnO modified paper-based ELISA (nano-p-ELISA). According to the verifying test using a collection of sera collected from brucellosis and non-brucellosis patients, the sensitivity and specificity of multiepitope based nano-p-ELISA were 92.38% and 98.35% respectively. The positive predictive value was 98.26% and the negative predictive value was 91.67%. The multiepitope based fusion protein also displayed significantly higher specificity than Brucella lipopolysaccharide (LPS) antigen. Conclusions B cell epitopes are important candidates for serologically testing brucellosis. Multiepitope fusion protein based nano-p-ELISA displayed significantly sensitivity and specificity compared to Brucella LPS antigen. The strategy applied in this study will be helpful to develop rapid and accurate diagnostic method for brucellosis in human as well as animal populations., Author summary Brucellosis is one of the most important zoonosis in the world and has caused tremendous economic losses in agriculture and animal husbandry in many countries. Developing rapid, sensitive and specific diagnostic methods is very important for early detection and treatment of brucellosis patients. In this study, a novel diagnostic technique, nano-ZnO modified paper ELISA, was established. The antigen used in this technique was a fusion protein containing multiple B cell epitopes, which were predicted from Brucella major outer membrane proteins such as Bp26, Omp31, Omp16, Omp2b and Omp25. Comparing to traditional LPS antigen, this multiepitope based antigen displayed considerably higher sensitivity and higher specificity in laboratory. With the strategy described in this paper, more efficient epitopes and protein antigen can be identified in the future. Currently, LPS antigen is only prepared from live Brucella, while protein antigen can be produced in large quantities in prokaryotic expression system. In addition to nano-p-ELISA, this protein antigen can also be used for development other methods such as fluorescent polarization assay (FPA) and immunochromatographic assay (ICA) to meet the varied demand for brucellosis testing.

Rabies is a lethal zoonotic encephalomyelitis that causes an estimated 59,000 human deaths yearly worldwide. Although developing countries of Asia and Africa bear the heaviest burden, surveillance and disease detection in these countries is often hampered by the absence of local laboratories able to diagnose rabies and/or the difficulties of sample shipment from low-access areas to national reference laboratories. Filter papers offer a convenient cost-effective alternative for the sampling, shipment, and storage of biological materials for the diagnosis of many pathogens including rabies virus, yet the properties of diagnostic tests using this support have not been evaluated thoroughly. Sensitivity and specificity of molecular diagnosis of rabies infection using a reverse transcription followed by a hemi-nested polymerase chain reaction (RT-hn-PCR) either directly on brain tissue or using brain tissue dried on filter paper were assessed on 113 suspected field animal samples in comparison to the direct fluorescent antibody test (FAT) recommended by the World Health Organization as one of the reference tests for rabies diagnosis. Impact of the duration of the storage was also evaluated. The sensitivity and the specificity of RT-hn-PCR i) on brain tissue were 96.6% (95% CI: [88.1–99.6]) and 92.7% (95% CI: [82.4–98.0]) respectively and ii) on brain tissue dried on filter paper 100% (95% CI: [93.8–100.0]) and 90.9% (95% CI: [80.0–97.0]) respectively. No loss of sensitivity of RT-hn-PCR on samples of brain tissue dried on filter paper left 7 days at ambient temperature was detected indicating that this method would enable analyzing impregnated filter papers sent to the national reference laboratory at ambient temperature within a 1-week shipment time. It could therefore be an effective alternative to facilitate storage and shipment of samples from low-access areas to enhance and expand rabies surveillance., Author summary Dogs are responsible for 99% of human rabies deaths. Facilitating diagnostic of rabid dogs can help further identify i) people who were exposed to rabies in order to encourage them to seek post exposure prophylaxis and ii) other exposed animals to break the transmission chain (i.e. prevent them from further transmitting the virus to other people and animals). Yet, the reference diagnostic method for animal rabies requires brain samples to be collected post-mortem and shipped under temperature-controlled conditions. The shipping of such samples is complex in low-access areas, especially in low income countries, which are often those that bear the heaviest rabies burden. Filter papers offer a convenient alternative for biological sample shipment and storage. Here the efficiency of a molecular diagnostic method applied to brain tissue dried on filter paper is compared to one of the reference methods, the direct fluorescent antibody test. Our results show it has an excellent sensitivity (it does not miss any positive samples), even when filter papers are left 7 days at ambient temperature. These results let us foresee a cost-effective alternative facilitating shipment, storage and testing of samples from rabies suspected animals from low-access areas. This could considerably enhance and expand rabies surveillance in low-income countries, allowing a more comprehensive evaluation of rabies burden, and thus reinforcing arguments for allocating funds to rabies control policies.

Background Global estimates for cholera annually approximate 4 million cases worldwide with 95,000 deaths. Recent outbreaks, including Haiti and Yemen, are reminders that cholera is still a global health concern. Cholera outbreaks can rapidly induce high death tolls by overwhelming the capacity of health facilities, especially in remote areas or areas of civil unrest. Recent studies demonstrated that stool specimens preserved on filter paper facilitate molecular analysis of Vibrio cholerae in resource limited settings. Specimens preserved in a rapid, low-cost, safe and sustainable manner for sequencing provides previously unavailable data about circulating cholera strains. This may ultimately contribute new information to shape public policy response on cholera control and elimination. Methodology/Principal findings Whole genome sequencing (WGS) recovered close to a complete sequence of the V. cholerae O1 genome with satisfactory genome coverage from stool specimens enriched in alkaline peptone water (APW) and V. cholerae culture isolates, both spotted on filter paper. The minimum concentration of V. cholerae DNA sufficient to produce quality genomic information was 0.02 ng/μL. The genomic data confirmed the presence or absence of genes of epidemiological interest, including cholera toxin and pilus loci. WGS identified a variety of diarrheal pathogens from APW-enriched specimen spotted filter paper, highlighting the potential for this technique to explore the gut microbiome, potentially identifying co-infections, which may impact the severity of disease. WGS demonstrated that these specimens fit within the current global cholera phylogenetic tree, identifying the strains as the 7th pandemic El Tor. Conclusions WGS results allowed for mapping of short reads from APW-enriched specimen and culture isolate spotted filter papers. This provided valuable molecular epidemiological sequence information on V. cholerae strains from remote, low-resource settings. These results identified the presence of co-infecting pathogens while providing rare insight into the specific V. cholerae strains causing outbreaks in cholera-endemic areas., Author summary Cholera affects more than 4 million people globally every year; people predominantly living in poverty or in resource-constrained conditions including political crises or natural disasters. Cholera’s typical presentation is characterized by rapid onset of acute watery diarrhea and vomiting which can progress from watery stool to shock in as little as four hours. Laboratory conditions needed for culture confirmation and strain preservation are rarely to never present in these affected areas. In fact, many cholera endemic areas in Sub-Saharan African are so remote that even treatment response alone is often challenging. Here we present the genomic analysis of DNA extracted from dried filter paper, which is a low-cost, low-tech and sustainable method. Previously this method has facilitated cholera confirmation by PCR, but we demonstrate that this method is also suitable for whole genome sequencing and subsequent strain characterization by presenting the analysis of samples from an outbreak in a remote area of Cameroon. This method will facilitate the understanding of the molecular epidemiology in cholera-prone areas, which were previously too challenging to attempt. It also introduces a method that can be used on a broader scale for diarrheal disease surveillance, including providing a window into co-infection and microbiome analyses.

Introduction: paper currency notes, exchangeable fomite, that is continuously contaminated because of the poor handling and storage practices. Objective: the general objective of the study was to determine the bacterial contamination of paper currency notes possessed by food vendors around Mulago National Referral Hospital Complex. Methods: a total of sixty paper notes of six denominations (1000, 2000, 5000, 10000, 20000, 50000) were collected from different food vendors. Each note was preserved in a sterile falcon tube and transported to the microbiology lab for bacteriological examination. Data from questionnaires was analyzed using SPSS version 23 (IBM SPSS Statistics). Results: all sampled paper notes had bacterial contamination. The bacterial counts ranged from 4×102 cfu/ml to 6.8×109 cfu/ml, with the Shs.1000 notes having the highest average total bacterial load of 2.17×109 cfu/ml and highest average total coli form counts of 21.5×102 cfu/ml. The fifty thousand shillings note had no coliform detected. Of the analysed 60 samples, 27(45%) samples contained Staphylococcus aureus. None of the sampled paper notes had Escherichia coli. Conclusion: the study revealed that most of Ugandan paper notes are contaminated with bacteria including potential pathogens that cause disease in healthy individuals and opportunistic pathogens that may cause disease in hospitalized and immunocompromised patients. This study showed that the most contaminated note denominations were those of low denomination (Shs.1000 and Shs.2000) which had the highest bacterial count. The study revealed the paper currency notes were stored in different places where the commonest was the drawer and kept with different items, the commonest being pens. Hence, great care must be taken while handling money during the preparation and handling of food to avoid cross contamination.

ObjectivesRecently, the results of two economic evaluations were published both of which seemingly demonstrate the cost-effectiveness of sofosbuvir-based regimens for the treatment of chronic hepatitis C genotype 1 infection in Germany. Both analyses were sponsored by the manufacturer of sofosbuvir and use a different methodology: Whereas one evaluation is based on a conventional cost-utility analysis, the other rests upon the efficiency-frontier method used by the German Institute for Quality and Efficiency in Health Care (IQWiG). The purpose of this study is to reanalysis the results of both economic evaluations in combination.DesignReanalysis of published decision modelling results.SettingPrimary care in Germany.ParticipantsPatients with chronic hepatitis C genotype 1 infection (treatment-naïve and -experienced, cirrhotic and non-cirrhotic).InterventionsSofosbuvir, other anti-hepatitis C virus drugs, and no treatment.Primary and secondary outcome measuresCost per unit of health benefit and cost per quality-adjusted life year.ResultsReanalysis of the results of both economic evaluations in combination reveals an unclear rationale for choosing the selected cost-effectiveness methods as well as a potential publication bias, favoring the product of the manufacturer. Based on the reanalysis, sofosbuvir is not cost-effective in treatment-experienced non-cirrhotic patients, potentially lacks cost-effectiveness in treatment-experienced cirrhotic patients, and is only partially cost-effective in treatment-naïve non-cirrhotic patients. Taken together, these results indicate a lack of cost-effectiveness in three quarters of the German patient population.ConclusionsTwo economic evaluations on sofosbuvir suggest, in combination, that sofosbuvir cannot be considered a cost-effective treatment in three quarters of the German patient population.

BACKGROUND The lack of information regarding the burden of acute bacterial meningitis in Latin America leads to a reduction in the estimated incidence rates of the disease, and impairs public health decisions on the use and follow-up of preventive interventions, particularly, the evaluation of existing vaccination policies. The use of the real-time PCR in diagnostic routine procedures has resulted in a substantial increase in confirmed bacterial meningitis cases. However, in resource-poor countries, these assays are only available in reference laboratories. Sample transportation to these laboratories is a critical constraint, as it requires specialized, high cost courier services. To overcome this barrier we evaluated the use of FTATM Elute filter paper cards for the conservation and processing of samples under normal environmental conditions, as they would be when transported from remote and under-equipped healthcare facilities to the reference centers. A total of 401 samples received in 2015 as part of Sao Paulo's national surveillance for routine diagnosis were selected for this study. METHODS The sensitivity and specificity of real-time PCR were evaluated using fresh serum and cerebrospinal fluid (CSF) samples processed using our laboratory's standard DNA extraction, and processing the same samples after being dried and stored on FTATM card, and DNA extracted following the manufacturer's instructions. RESULTS The sensitivities for detection of Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae from CSF dried and stored on FTATM cards were 98%, 92%, and 100%, respectively, and with serum samples were 73%, 88%, and 100%, respectively. When compared to our laboratory's standard methodology, results showed high concordance, with Kappa index ranges of 0.9877-1.00 for CSF, and 0.8004-1.00 for serum samples. CONCLUSION The use of FTATM cards for CSF and serum conservation and transport represents a rapid, reliable, and cost-effective alternative that will allow obtaining valuable epidemiological information that would otherwise be lost.

International audience; Scenario analysis constitutes a useful approach to synthesize knowledge and derive hypotheses in the case of complex systems that are documented with mainly qualitative or very diverse information. In this article, a framework for scenario analysis is designed and then, applied to global wheat health within a timeframe from today to 2050. Scenario analysis entails the choice of settings, the definition of scenarios of change, and the analysis of outcomes of these scenarios in the chosen settings. Three idealized agrosystems, representing a large fraction of the global diversity of wheat-based agrosystems, are considered, which represent the settings of the analysis. Several components of global changes are considered in their consequences on global wheat health: climate change and climate variability, nitrogen fertilizer use, tillage, crop rotation, pesticide use, and the deployment of host plant resistances. Each idealized agrosystem is associated with a scenario of change that considers first, a production situation and its dynamics, and second, the impacts of the evolving production situation on the evolution of crop health. Crop health is represented by six functional groups of wheat pathogens: the pathogens associated with Fusarium head blight; biotrophic fungi, Septoria-like fungi, necrotrophic fungi, soilborne pathogens, and insect-transmitted viruses. The analysis of scenario outcomes is conducted along a risk-analytical pattern, which involves risk probabilities represented by categorized probability levels of disease epidemics, and risk magnitudes represented by categorized levels of crop losses resulting from these levels of epidemics within each production situation. The results from this scenario analysis suggest an overall increase of risk probabilities and magnitudes in the three idealized agrosystems. Changes in risk probability or magnitude however vary with the agrosystem and the functional groups of pathogens. We discuss the effects of global changes on the six functional groups, in terms of their epidemiology and of the crop losses they cause. Scenario analysis enables qualitative analysis of complex systems, such as plant pathosystems that are evolving in response to global changes, including climate change and technology shifts. It also provides a useful framework for quantitative simulation modeling analysis for plant disease epidemiology.

We propose to use Twitter data as social-spatial sensors. This study deals with the question whether research papers on certain diseases are perceived by people in regions (worldwide) that are especially concerned by these diseases. Since (some) Twitter data contain location information, it is possible to spatially map the activity of Twitter users referring to certain papers (e.g., dealing with tuberculosis). The resulting maps reveal whether heavy activity on Twitter is correlated with large numbers of people having certain diseases. In this study, we focus on tuberculosis, human immunodeficiency virus (HIV), and malaria, since the World Health Organization ranks these diseases as the top three causes of death worldwide by a single infectious agent. The results of the social-spatial Twitter maps (and additionally performed regression models) reveal the usefulness of the proposed sensor approach. One receives an impression of how research papers on the diseases have been perceived by people in regions that are especially concerned by these diseases. Our study demonstrates a promising approach for using Twitter data for research evaluation purposes beyond simple counting of tweets.

Background The use of filter paper as a simple, inexpensive tool for storage and transportation of blood, ‘Dried Blood Spots’ or Guthrie cards, for diagnostic assays is well-established. In contrast, there are a paucity of diagnostic evaluations of dried cerebrospinal fluid (CSF) spots. These have potential applications in low-resource settings, such as Laos, where laboratory facilities for central nervous system (CNS) diagnostics are only available in Vientiane. In Laos, a major cause of CNS infection is Japanese encephalitis virus (JEV). We aimed to develop a dried CSF spot protocol and to evaluate its diagnostic performance using the World Health Organisation recommended anti-JEV IgM antibody capture enzyme-linked immunosorbent assay (JEV MAC-ELISA). Methodology and Principal Findings Sample volumes, spotting techniques and filter paper type were evaluated using a CSF-substitute of anti-JEV IgM positive serum diluted in Phosphate Buffer Solution (PBS) to end-limits of detection by JEV MAC-ELISA. A conventional protocol, involving eluting one paper punch in 200μl PBS, did not detect the end-dilution, nor did multiple punches utilising diverse spotting techniques. However, pre-cut filter paper enabled saturation with five times the volume of CSF-substitute, sufficiently improving sensitivity to detect the end-dilution. The diagnostic accuracy of this optimised protocol was compared with routine, neat CSF in a pilot, retrospective study of JEV MAC-ELISA on consecutive CSF samples, collected 2009–15, from three Lao hospitals. In comparison to neat CSF, 132 CSF samples stored as dried CSF spots for one month at 25–30°C showed 81.6% (65.7–92.3 95%CI) positive agreement, 96.8% (91.0–99.3 95%CI) negative agreement, with a kappa coefficient of 0.81 (0.70–0.92 95%CI). Conclusions/Significance The novel design of pre-cut filter paper saturated with CSF could provide a useful tool for JEV diagnostics in settings with limited laboratory access. It has the potential to improve national JEV surveillance and inform vaccination policies. The saturation of filter paper has potential use in the wider context of pathogen detection, including dried spots for detecting other analytes in CSF, and other body fluids., Author Summary Japanese encephalitis virus infection is widespread in Asia, and primarily affects children in poor, rural areas. The virus spreads to the brain and spinal cord resulting in significant death and disability. Diagnosis requires testing for the immune response ‘antibody’ specific to Japanese encephalitis virus in the cerebrospinal fluid that surrounds the brain and spinal cord. However, in most areas where the infection occurs there are no laboratory facilities. In the absence of laboratory diagnosis, we have poor knowledge of the extent of the problem in these areas. Dried blood spots on filter paper have been used widely for many years as simple, cost-effective tools for transporting samples for testing. However, there have been few attempts at using dried cerebrospinal fluid spots. We developed a technique for using dried spots to store and test cerebrospinal fluid for antibodies to Japanese encephalitis virus. We compared the results of dried spots with routine neat cerebrospinal fluid in 132 patient samples, and demonstrated excellent agreement between the two tests. This novel method for saturating dried cerebrospinal fluid spots has the potential to enhance our knowledge of Japanese encephalitis virus epidemiology, and inform health policies where they are most needed. It could also be transferred for use in diagnosing other infectious diseases, including using other body fluid samples.

Pyocyanin is a toxin produced by Pseudomonas aeruginosa. Here we describe a novel paper-based electrochemical sensor for pyocyanin detection, manufactured with a simple and inexpensive approach based on electrode printing on paper. The resulting sensors constitute an effective electrochemical method to quantify pyocyanin in bacterial cultures without the conventional time consuming pretreatment of the samples. The electrochemical properties of the paper-based sensors were evaluated by ferri/ferrocyanide as a redox mediator, and showed reliable sensing performance. The paper-based sensors readily allow for the determination of pyocyanin in bacterial cultures with high reproducibility, achieving a limit of detection of 95 nM and a sensitivity of 4.30 μA/μM in standard culture media. Compared to the similar commercial ceramic based sensors, it is a 2.3-fold enhanced performance. The simple in-house fabrication of sensors for pyocyanin quantification allows researchers to understand in vitro adaptation of P. aeruginosa infections via rapid screenings of bacterial cultures that otherwise are expensive and time-consuming.

Biomaterial-implants are frequently used to restore function and form of human anatomy. However, the presence of implanted biomaterials dramatically elevates infection risk. Paradoxically, dental-implants placed in a bacteria-laden milieu experience moderate failure-rates, due to infection (0.0-1.1 %), similar to the ones of joint-arthroplasties placed in a near-sterile environment (0.1-1.3 %). Transcutaneous bone-fixation pins breach the immune-barrier of the epidermis, exposing underlying sterile-tissue to an unsterile external environment. In contrast to dental-implants, also placed in a highly unsterile environment, these pins give rise to relatively high infection-associated failure-rates of up to 23.0 %. Herein, we attempt to identify causes as to why dental-implants so often succeed, where others fail. The major part of all implants considered are metal-made, with similar surface-finishes. Material choice was therefore discarded as underlying the paradox. Antimicrobial activity of saliva has also been suggested as a cause for the success of dental-implants, but was discarded because saliva is the implant-site-fluid from which viable bacteria adhere. Crevicular fluid was discarded as it is largely analogous to serum. Instead, we attribute the relative success of dental-implants to (1) ability of oral tissues to heal rapidly in the continuous presence of commensal bacteria and opportunistic pathogens, and (2) tolerance of the oral immune-system. Inability of local tissue to adhere, spread and grow in presence of bacteria and an intolerant immune-system are identified as the likely main causes explaining the susceptibility of other implants to infection-associated failure. In conclusion, it is the authors’ belief that new anti-infection strategies for a wide range of biomaterial-implants may be derived from the relative success of dental-implants.

This study aimed to investigate the co-composting time effectiveness as well as the effect of the initial Carbon/Nitrogen ratio (C/N)i variation on the hygienization of olive pomace and turkey manure. Six different heaps, at 3 levels of (C/N)i ratios: 20, 22 and 28, were installed and monitored during 6 months and assessed at three steps: At the beginning, the end of thermophilic-phase and the end of curing-phase. The microbial monitoring concerned 5 microbial pathogens contents, used as hygiene microbial indicators, namely: Sulphite-Reducing Anaerobes (SRA), Escherichia Coli (E. Coli), Total Aerobic Mesophilic Flora (TAMF), Staphylococci, and Salmonella spp. Initially, the mixtures showed high TAMF and Staphylococci loads. Meanwhile, SRA and E. coli populations were relatively low and Salmonella spp. was not detected. The microbial assessment showed a significant effect of composting time on the reduction of pathogens load, except for SRA where its population has increased significantly, while the (C/N)i had a non-significant effect on pathogen content of the end-product. The final values expressed as colony-forming unit per gram (CFU g-1), were as follow: Sulfite-reducing Anaerobes (≤3.1 x 103 CFU g-1), E. Coli germ used as an indicator of faecal contamination (

Infectious diseases are potential drivers for human evolution, through a complex, continuous and dynamic interaction between the host and the pathogen/s. It is this dynamic interaction that contributes toward the clinical outcome of a pathogenic disease. These are modulated by contributions from the human genetic variants, transcriptional response (including noncoding RNA) and the pathogen’s genome architecture. Modern genomic tools and techniques have been crucial for the detection and genomic characterization of pathogens with respect to the emerging infectious diseases. Aided by next-generation sequencing (NGS), risk stratification of host population/s allows for the identification of susceptible subgroups and better disease management. Nevertheless, many challenges to a general understanding of host–pathogen interactions remain. In this review, we elucidate how a better understanding of the human host-pathogen interplay can substantially enhance, and in turn benefit from, current and future applications of multi-omics based approaches in infectious and rare diseases. This includes the RNA-level response, which modulates the disease severity and outcome. The need to understand the role of human genetic variants in disease severity and clinical outcome has been further highlighted during the Coronavirus disease 2019 (COVID-19) pandemic. This would enhance and contribute toward our future pandemic preparedness.

International audience; Mosquito control is implemented when arboviruses are detected in patients or in field-collected mosquitoes. However, mass screening of mosquitoes is usually laborious and expensive, requiring specialized expertise and equipment. Detection of virus in mosquito saliva using honey-impregnated filter papers seems to be a promising method as it is non-destructive and allows monitoring the viral excretion dynamics over time from the same mosquito. Here we test the use of filter papers to detect chikungunya virus in mosquito saliva in laboratory conditions, before proposing this method in large-scale mosquito surveillance programs. We found that 0.9 cm 2 cards impregnated with a 50% honey solution could replace the forced salivation technique as they offered a viral RNA detection until 7 days after oral infection of Aedes aegypti and Aedes albopictus mosquitoes with CHIKV.

Soil physicochemical properties and fungal communities are pivotal factors for continuous cropping of American ginseng (Panax quinquefoliumL.). However, the response of soil physicochemical properties and fungal communities to replant disease of American ginseng has not yet been studied. High-throughput sequencing and soil physicochemical analyses were undertaken to investigate the difference of soil fungal communities and environmental driver factors in new and old ginseng fields; the extent of replant disease in old ginseng fields closely related to changes in soil properties and fungal communities was also determined. Results indicated that fungal communities in an old ginseng field were more sensitive to the soil environment than those in a new ginseng field, and fungal communities were mainly driven by soil organic matter (SOM), soil available phosphorus (AP), and available potassium (AK). Notably, healthy ginseng plants in new and old ginseng fields may influence fungal communities by actively recruiting potential disease suppressive fungal agents such asAmphinema,Cladophialophora,Cadophora,Mortierella,andWilcoxina. When these key groups and members were depleted, suppressive agents in the soil possibly declined, increasing the abundance of pathogens. Soil used to grow American ginseng in the old ginseng field contained a variety of fungal pathogens, includingAlternaria,Armillaria,Aphanoascus,Aspergillus,Setophoma,andRhexocercosporidium. Additionally, micro-ecological factors affecting disease outbreaks in the old ginseng field included a strengthening in competition relationships, a weakening in cooperation relationships, and a change of trophic strategies among fungal communities.

The highly contagious nature of SARS-CoV-2 has led to several studies on the transmission of the virus. A little studied potential fomite of great concern in the community is currency, which has been shown to harbor microbial pathogens in several studies. Since the onset of the COVID-19 pandemic, many businesses in the United States have limited the use of banknotes in favor of credit cards. However, SARS-CoV-2 has shown greater stability on plastic in several studies. Herein, the stability of SARS-CoV-2 at room temperature on banknotes, money cards and coins was investigated. In vitro studies with live virus suggested SARS-CoV-2 was highly unstable on banknotes, showing an initial rapid reduction in viable virus and no viral detection by 24 hours. In contrast, SARS-CoV-2 displayed increased stability on money cards with live virus detected after 48 hours. Environmental swabbing of currency and money cards on and near the campus of Brigham Young University supported these results, with no detection of SARS-CoV-2 RNA on banknotes, and a low level on money cards. However, no viable virus was detected on either. These preliminary results suggest that the use of money cards over banknotes in order to slow the spread of this virus may be ill-advised. These findings should be investigated further through larger environmental studies involving more locations.

Zoonotic diseases—human diseases of animal origin—represent one of the world's greatest health challenges, both today and in the past. Since the Neolithic, zoonotic diseases have been one of the major factors shaping and influencing human adaptation. Archaeology is ideally situated to provide the long view on human–animal–pathogen relationships through combining cultural, environmental and biological datasets, yet long‐term studies of linked human and animal records have often been overlooked and undertheorized. The seven papers in this special issue “Zoonotic diseases: New directions in human–animal pathology” cover a range of diseases caused by bacterial, viral, and parasitic pathogens, from case studies drawn from across Europe, Asia, Africa and the Americas. They speak to the diversity of human–animal–environment interactions that shaped disease emergence and transmission. They also review methodological advancements relating to disease identification and interpretation and discuss interdisciplinary approaches to effectively investigate these complex processes in the past. This introduction highlights their key themes and outcomes and identifies research priorities moving forward.

Given the increased reporting of multi-resistant bacteria and the shortage of newly approved medicines, researchers have been looking towards extreme and unusual environments as a new source of antibiotics. Streptomyces currently provides many of the world’s clinical antibiotics, so it comes as no surprise that these bacteria have recently been isolated from traditional medicine. Given the wide array of traditional medicines, it is hoped that these discoveries can provide the much sought after core structure diversity that will be required of a new generation of antibiotics. This review discusses the contribution of Streptomyces to antibiotics and the potential of newly discovered species in traditional medicine. We also explore how knowledge of traditional medicines can aid current initiatives in sourcing new and chemically diverse antibiotics.

Wastewater sludge is a by-product of waste water treatment; it is often employed in agricultural processes following appropriate microbiological stabilization. In this study, we evaluated two methods of sludge stabilization in the waste water treatment plant of Sotaquirá, Colombia. The two assessed stabilization methods were dewatering on drying beds and alkaline stabilization. The stabilization through dewatering on drying bed was carried out for five months. Alkaline stabilization was conducted for 96 hours with three concentrations of calcium oxide, 7%, 9%, and 13% w/w. Humidity, pH, organic carbon, phosphorus, organic nitrogen, fecal coliforms, somatic phages, total helminth eggs, and Salmonella sp. were monitored monthly for the sludge under dewatering treatment and at 0, 12, 24 hours for sludge under alkaline stabilization treatment. Both treatments succeded in eliminating Salmonella sp. Helminth eggs were eliminated by alkaline stabilization, whereas it was reduced to one or zero helminth eggs with the dewatering treatment. Somatic phages were eliminated with alkaline stabilization but were only reduced to 3.52 log CFU/g with the dewatering method. Dewatering on drying beds produced biosolids that can be used for soil restoration. Whereas alkaline stabilization produced biosolids that can be used for agricultural purposes. Alkaline stabilization with 9% and 13 % calcium oxide ostensibly reduced nitrogen and phosphorus contents in the sludge, whereas 7 % calcium affected less the phosphorus concentration of the sludge. These results indicate that sludge dewatering on drying beds is an effective sludge sanitation protocol to be implemented in small waste water treatment plants, such that in Sotaquirá, Colombia.

Background: Currency notes represent a universal medium for transmission of bacteria in the environment and among humans. Care should be taken by those handling these currencies. Aim: To quantitatively assess and compare the total bacterial load of Staphylococcus aureus, Escherichia coli, and Proteus species present on the currency notes circulating in the open market area and hospitals in Mangalore city. Methodology: A total of 24 frequently circulated currency notes were collected from open markets and hospitals (12 from each group). They were then deposited in sterile pouches. Samples were collected using moistened cotton swabs and inoculated onto MacConkey agar for bacterial growth. The isolated bacteria were then assessed for the percentage of bacteria and colony-forming units (CFUs) on the notes using standardized microbial analysis. Results: Cultures after microbial analysis showed bacterial contamination with S. aureus, E. coli, Klebsiella, and aerobic spore-bearing bacteria. Approximately >103 CFUs/plate were found. In the hospital group, isolated organisms comprised S. aureus (54%) and E. coli(25%); in the open market areas, E. coli(36%) and S. aureus (38%) were found. Proteus species were not present on any of the samples. Intergroup statistical analysis carried out using the Chi-square test was statistically significant (P = 0.034). Conclusion: The study concluded that the notes from hospitals were contaminated more with S.aureus than in open markets, whereas E. coli was found to be more in open market areas than in hospitals.

Graphical abstract, The global outbreak of coronavirus infectious disease-2019 (COVID-19) draws attentions in the transport and spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in aerosols, wastewater, surface water and solid wastes. As pathogens eventually enter the subsurface system, e.g., soils in the vadose zone and groundwater in the aquifers, they might survive for a prolonged period of time owing to the uniqueness of subsurface environment. In addition, pathogens can transport in groundwater and contaminate surrounding drinking water sources, possessing long-term and concealed risks to human society. This work critically reviews the influential factors of pathogen migration, unravelling the impacts of pathogenic characteristics, vadose zone physiochemical properties and hydrological variables on the migration of typical pathogens in subsurface system. An assessment algorithm and two rating/weighting schemes are proposed to evaluate the migration abilities and risks of pathogens in subsurface environment. As there is still no evidence about the presence and distribution of SARS-CoV-2 in the vadose zones and aquifers, this study also discusses the migration potential and behavior of SARS-CoV-2 viruses in subsurface environment, offering prospective clues and suggestions for its potential risks in drinking water and effective prevention and control from hydrogeological points of view.

Many microbial phenotypes are differentially or exclusively expressed on agar surfaces, including biofilms, motility, and sociality. However, agar-based assays are limited by their low throughput, which increases costs, lab waste, space requirements, and the time required to conduct experiments. Here, we demonstrate the use of wax-printed microfluidic paper-based analytical devices (μPADs) to measure linear growth rate of microbes on an agar growth media as a means of circumventing the aforementioned limitations. The main production materials of the proposed μPAD design are a wax printer, filter paper, and empty pipet boxes. A single wax-printed μPAD allowing 8 independent, agar-grown colonies costs $0.07, compared to $0.20 and $9.37 for the same number of replicates on traditional microtiter/spectrophotometry and Petri dish assays, respectively. We optimized the μPAD design for channel width (3 mm), agar volume (780 μL/channel), and microbe inoculation method (razor-blade). Comparative analyses of the traditional and proposed μPAD methods for measuring growth rate of nonmotile (Saccharomyces cerevisiae) and motile (flagellated Escherichia coli) microorganisms suggested the μPAD assays conferred a comparable degree of accuracy and reliability to growth rate measurements as their traditional counterparts. We substantiated this claim with strong, positive correlations between the traditional and μPAD assay, a significant nonzero slope in the model relating the two assays, a nonsignificant difference between the relative standard errors of the two techniques, and an analysis of inter-device reliability. Therefore, μPAD designs merit consideration for the development of enhanced-throughput, low-cost microbial growth and motility assays.

Laundering of textiles—clothing, linens, and cleaning cloths—functionally removes dirt and bodily fluids, which prevents the transmission of and reexposure to pathogens as well as providing odor control. Thus, proper laundering is key to controlling microbes that cause illness and produce odors. The practice of laundering varies from region to region and is influenced by culture and resources. This review aims to define laundering as a series of steps that influence the exposure of the person processing the laundry to pathogens, with respect to the removal and control of pathogens and odor-causing bacteria, while taking into consideration the types of textiles. Defining laundering in this manner will help better educate the consumer and highlight areas where more research is needed and how to maximize products and resources. The control of microorganisms during laundering involves mechanical (agitation and soaking), chemical (detergent and bleach), and physical (detergent and temperature) processes. Temperature plays the most important role in terms of pathogen control, requiring temperatures exceeding 40°C to 60°C for proper inactivation, while detergents play a role in reducing the microbial load of laundering through the release of microbes attached to fabrics and the inactivation of microbes sensitive to detergents (e.g., enveloped viruses). The use of additives (enzymes) and bleach (chlorine and activated oxygen) becomes essential in washes with temperatures below 20°C, especially for certain enteric viruses and bacteria. A structured approach is needed that identifies all the steps in the laundering process and attempts to identify each step relative to its importance to infection risk and odor production.

Among the ultimate requirements of biosensor technology, sensitivity, stability, and specificity are undoubtedly crucial features to enter the market. Actually, sensitivity is a major drawback for biosensors, especially where there is a low abundance of targets. To address this concern, biosensor technology has been supported by effective methodologies for the target isothermal amplification, successfully applied to sensitively detect nucleic acids of viral and bacterial sources, capable to operate in low-resource settings and instrumentations, an important prerequisite for microfluidics and point-of-care diagnostics. This review of 133 refs provides an in-depth description of the available methods for isothermal amplification of nucleic acids, with pros and cons, and their integration into different detection systems. The potential of combining the amplification-powered biosensor with technological advances in the branches of nanotechnology and paper microfluidics is also proved by excellent examples from the literature.

In this paper we analyse the feasibility of low voltage iron electrocoagulation as a means of municipal secondary effluent treatment with a focus on removal of microbial indicators, Antibiotic Resistant Bacteria (ARB) and nutrients. A laboratory scale batch unit equipped with iron electrodes was used on synthetic and real secondary effluent from a municipal wastewater treatment plant. Synthetic secondary effluent was separately assayed with spiked Escherichia coli WR1 and with bacteriophage ΦX174, while real effluent samples were screened before and after treatment for E. coli, Extended Spectrum Betalactamase-producing E. coli, Enterococci, Vancomycin Resistant Enterococci, Clostridium perfringens spores and somatic coliphages. Charge dosage (CD) and charge dosage rate (CDR) were used as the main process control parameters. Experiments with synthetic secondary effluent showed >4log10 and >5log10 removal for phage ΦX174 and for E. coli WR1, respectively. In real effluents, bacterial indicator removal exceeded 3.5log10, ARB were removed below detection limit (≥2.5log10), virus removal reached 2.3log10 and C. perfringens spore removal exceeded 2.5log10. Experiments in both real and synthetic wastewater showed that bacterial removal increased with increasing CD and decreasing CDR. Virus removal increased with increasing CD but was irresponsive to CDR. C. perfringens spore removal increased with increasing CD yet reached a removal plateau, being also irresponsive to CDR. Phosphate removal exceeded 99%, while total nitrogen and chemical oxygen demand removal were below 15% and 58%, respectively. Operational cost estimates were made for power and iron plate consumption, and were found to be in the range of 0.01 to 0.24€/m3 for the different assayed configurations. In conclusion, low voltage Fe-EC is a promising technology for pathogen reduction of secondary municipal effluents, with log10 removals comparable to those achieved by conventional disinfection methods such as chlorination, UV or ozonation.

In 2017, the 560-ha area of hybrid poplar plantation in northern Poland showed symptoms of tree decline. Leaves appeared smaller, turned yellow-brown, and were shed prematurely. Twigs and smaller branches died. Bark was sunken and discolored, often loosened and split. Trunks decayed from the base. Phloem and xylem showed brown necrosis. Ten per cent of trees died in 1–2 months. None of these symptoms was typical for known poplar diseases. Bacteria in soil and the necrotic base of poplar trunk were analysed with Illumina sequencing. Soil and wood were colonized by at least 615 and 249 taxa. The majority of bacteria were common to soil and wood. The most common taxa in soil were: Acidobacteria (14.757%), Actinobacteria (14.583%), Proteobacteria (36.872) with Betaproteobacteria (6.516%), Burkholderiales (6.102%), Comamonadaceae (2.786%), and Verrucomicrobia (5.307%).The most common taxa in wood were: Bacteroidetes (22.722%) including Chryseobacterium (5.074%), Flavobacteriales (10.873%), Sphingobacteriales (9.396%) with Pedobacter cryoconitis (7.306%), Proteobacteria (73.785%) with Enterobacteriales (33.247%) including Serratia (15.303%) and Sodalis (6.524%), Pseudomonadales (9.829%) including Pseudomonas (9.017%), Rhizobiales (6.826%), Sphingomonadales (5.646%), and Xanthomonadales (11.194%). Possible pathogens were Pseudomonas, Rhizobium and Xanthomonas. The initially endophytic character of bacteria is emphasized. Soil and possibly planting material might be the sources of pathogen inoculum.

The aerobic granular sludge (AGS) process is an effective wastewater treatment technology for organic matter and nutrient removal that has been introduced in the market rapidly. Until now, limited information is available on AGS regarding the removal of bacterial and viral pathogenic organisms present in sewage. This study focussed on determining the relation between reactor operational conditions (plug flow feeding, turbulent aeration and settling) and physical and biological mechanisms on removing two faecal surrogates, Escherichia coli and MS2 bacteriophages. Two AGS laboratory-scale systems were separately fed with influent spiked with 1.0 × 106 CFU/100 mL of E. coli and 1.3 × 108 PFU/100 mL of MS2 bacteriophages and followed during the different operational phases. The reactors contained only granular sludge and no flocculent sludge. Both systems showed reductions in the liquid phase of 0.3 Log10 during anaerobic feeding caused by a dilution factor and attachment of the organisms on the granules. Higher removal efficiencies were achieved during aeration, approximately 1 Log10 for E. coli and 0.6 Log10 for the MS2 bacteriophages caused mainly by predation. The 18S sequencing analysis revealed high operational taxonomic units (OTUs) of free-living protozoa genera Rhogostoma and Telotrochidium concerning the whole eukaryotic community. Attached ciliates propagated after the addition of the E. coli, an active contribution of the genera Epistylis, Vorticella, and Pseudovorticella was found when the reactor reached stability. In contrast, no significant growth of predators occurred when spiking the system with MS2 bacteriophages, indicating a low contribution of protozoa on the phage removal. Settling did not contribute to the removal of the studied bacterial and viral surrogates. M.L. Barrios-Hernández acknowledges the Technological Institute of Costa Rica for providing the fellowship (Grant Number 007-2014-M) to pursue her PhD programme (2016-2020) at IHE-Delft, the Netherlands. K. Mora-Cabrera acknowledges the Generalitat Valenciana (GRISOLIAP/2017/173) and the European Social Funds (BEFPI/2019/065) for their financial support.

Calcium signaling can elicit different pathways involved in an extreme variety of biological processes. Calcium levels must be tightly regulated in a spatial and temporal manner in order to be efficiently and properly utilized in the host physiology. The Ca2+-ATPase, encoded by pmr-1 gene, was first identified in yeast and localized to the Golgi and it appears to be involved in calcium homeostasis. PMR-1 function is evolutionary conserved from yeast to human, where mutations in the orthologous gene ATP2C1 cause Hailey-Hailey disease. In this work, we used the Caenorhabditis elegans model system to gain insight into the downstream response elicited by the loss of pmr-1 gene. We found that pmr-1 knocked down animals not only showed defects in the oligosaccharide structure of glycoproteins at the cell surface but also were characterized by reduced susceptibility to bacterial infection. Although increased resistance to the infection might be related to lack of regular recognition of C. elegans surface glycoproteins by microbial agents, we provide genetic evidence that pmr-1 interfered nematodes mounted a stronger innate immune response to Gram-positive bacterial infection. Thus, our observations indicate pmr-1 as a candidate gene implicated in mediating the worm’s innate immune response.

Context: Nigerian soil fungi population is unexplored. It is hypothesized that they harbour new bioactive chemicals. This hypothesis is based on the large percentage of currently approved medicines that originated from soil-inhabiting micro-organisms Objectives: To investigate the antimicrobial and anti-inflammatory properties of Fusarium solani ethyl acetate (EtOAc) extract selected based on its broad spectrum of antimicrobial potential in an overlay experiment with seven other soil fungi strains. Materials and methods: Fungus number 6 (F6), identified by molecular characterization as Fusarium solani (Mart.) Sacc (Nectriaceae) was selected for studies from eight purified soil fungi due to its superior broad-spectrum antibiotics producing potential following agar overlay experiment. F6 was fermented for 21 d and the minimum inhibitory concentration (MIC) of its EtOAc fermentation extract (dose range: 12.5–100 µg/mL) was determined using agar dilution method for Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli, Salmonella typhi and anti-inflammatory properties determined using rat-paw (250–500 mg/kg) and xylene induced oedema (250–500 µg/kg) (in Swiss albino rats and mice) models, respectively. The ability of the extract to inhibit cyclooxygenase (COX) enzyme was also determined in vitro using Cayman test kit-760111. Result: The MIC of the EtOAc extract was

Sewage sludge is a by-product in the wastewater treatment and is an inherent hazardous issue because of the pathogenic contamination of natural resources. Therefore, in this study, domestic sludge was treated with premontane forest soil, macronutrients, and also pasteurization to reduce the content of volatile solids and pathogens. The best biostabilization treatment using premontane forest soil and pasteurization obtained a volatile solids reduction of 87% according to the environmental regulations, in which a biosolid is stable in a range of 38% of volatile solids reduction. In less than 30 days in a mesophilic range, the coliform count was reduced up to 71% when using forest soil and pasteurization. Thus, a biosolid-class B was obtained using gravimetric means as a platform to promote fast quality control.

Despite the development of effective vaccines against SARS-CoV-2, epidemiological control of the virus is still challenging due to slow vaccine rollouts, incomplete vaccine protection to current and emerging variants, and unwillingness to get vaccinated. Therefore, frequent testing of individuals to identify early SARS-CoV-2 infections, contact-tracing and isolation strategies remain crucial to mitigate viral spread. Here, we describe WHotLAMP, a rapid molecular test to detect SARS-CoV-2 in saliva. WHotLAMP is simple to use, highly sensitive (3.6 viral RNA copies per microliter of saliva) and specific, as well as inexpensive, making it ideal for frequent screening. Moreover, WHotLAMP does not require toxic chemicals or specialized equipment and thus can be performed in point-of-care settings, and may also be adapted for resource-limited environments or home use. While applied here to SARS-CoV-2, WHotLAMP can be modified to detect other pathogens, making it adaptable for other diagnostic assays, including for use in future outbreaks.

Summary There is strong evidence for a phylogenetic signal in the degree to which species share co‐evolved biotic partners and in the outcomes of biotic interactions. This implies there should be a phylogenetic signal in the outcome of feedbacks between plants and the soil microbiota they cultivate. However, attempts to identify a phylogenetic signal in plant–soil feedbacks have produced mixed results.Here we clarify how phylogenetic signals could arise in plant–soil feedbacks and use a recent compilation of data from feedback experiments to identify: whether there is a phylogenetic signal in the outcome of plant–soil feedbacks; and whether any signal arises through directional or divergent changes in feedback outcomes with evolutionary time.We find strong evidence for a divergent phylogenetic signal in feedback outcomes. Distantly related plant species show more divergent responses to each other’s soil microbiota compared with closely related plant species. The pattern of divergence implies occasional co‐evolutionary shifts in how plants interact with soil microbiota, with strongly contrasting feedback responses among some plant lineages.Our results highlight that it is difficult to predict feedback outcomes from phylogeny alone, other than to say that more closely related species tend to have more similar responses.

Colletotrichum lentis causes anthracnose, which is a serious disease on lentil and can account for up to 70% crop loss. Two pathogenic races, 0 and 1, have been described in the C. lentis population from lentil. To unravel the genetic control of virulence, an isolate of the virulent race 0 was sequenced at 1481‐fold genomic coverage. The 56.10‐Mb genome assembly consists of 50 scaffolds with N 50 scaffold length of 4.89 Mb. A total of 11 436 protein‐coding gene models was predicted in the genome with 237 coding candidate effectors, 43 secondary metabolite biosynthetic enzymes and 229 carbohydrate‐active enzymes (CAZymes), suggesting a contraction of the virulence gene repertoire in C. lentis. Scaffolds were assigned to 10 core and two minichromosomes using a population (race 0 × race 1, n = 94 progeny isolates) sequencing‐based, high‐density (14 312 single nucleotide polymorphisms) genetic map. Composite interval mapping revealed a single quantitative trait locus (QTL), qClVIR‐11, located on minichromosome 11, explaining 85% of the variability in virulence of the C. lentis population. The QTL covers a physical distance of 0.84 Mb with 98 genes, including seven candidate effector and two secondary metabolite genes. Taken together, the study provides genetic and physical evidence for the existence of a minichromosome controlling the C. lentis virulence on lentil.

The use of personal protective equipment (PPE) has been considered the most effective way to avoid the contamination of healthcare workers by different microorganisms, including SARS-CoV-2. A spray disinfection technology (chamber) was developed, and its efficacy in instant decontamination of previously contaminated surfaces was evaluated in two exposure times. Seven test microorganisms were prepared and inoculated on the surface of seven types of PPE (respirator mask, face shield, shoe, glove, cap, safety glasses and lab coat). The tests were performed on previously contaminated PPE using a manikin with a motion device for exposure to the chamber with biocidal agent (sodium hypochlorite) for 10 and 30s. In 96.93% of the experimental conditions analyzed, the percentage reduction was >99% (the number of viable cells found on the surface ranged from 4.3x106 to E. faecalis collected from the glove showed the lowest percentages reduction, with 86.000 and 86.500% for exposure times of 10 and 30 s, respectively. The log10 reduction values varied between 0.85 log10 (E. faecalis at 30 s in glove surface) and 9.69 log10 (E. coli at 10 and 30 s in lab coat surface). In general, E. coli, S. aureus, C. freundii, P. mirabilis, C. albicans and C. parapsilosis showed susceptibility to the biocidal agent under the tested conditions, with >99% reduction after 10 and 30s, while E. faecalis and P. aeruginosa showed a lower susceptibility. The 30s exposure time was more effective for the inactivation of the tested microorganisms. The results show that the spray disinfection technology has the potential for instant decontamination of PPE, which can contribute to an additional barrier for infection control of healthcare workers in the hospital environment.

International audience; The widespread use of facemasks throughout the population is recommended by the WHO to reduce transmission of the SARS-CoV-2 virus. As some regions of the world are facing mask shortages, reuse may be necessary. However, used masks are considered as a potential hazard that may spread and transmit disease if they are not decontaminated correctly and systematically before reuse. As a result, the inappropriate decontamination practices that are commonly witnessed in the general public are challenging management of the epidemic at a large scale. To achieve public acceptance and implementation, decontamination procedures need to be low-cost and simple. We propose the use of hot hygroscopic materials to decontaminate non-medical facemasks in household settings. We report on the inactivation of a viral load on a facial mask exposed to hot hygroscopic materials for 15 minutes. As opposed to recent academic studies whereby decontamination is achieved by maintaining heat and humidity above a given value, a more flexible procedure is proposed here using a slow decaying pattern, which is both effective and easier to implement, suggesting straightforward public deployment and hence reliable implementation by the population.

The main animal reservoirs of zoonotic hepatitis E virus (HEV) are domestic pigs and wild boars, but HEV also infects cervids. In this study, we estimated the prevalence of HEV in Finnish cervid species that are commonly hunted for human consumption. We investigated sera from 342 European moose (Alces alces), 70 white-tailed deer (Odocoileus virginianus), and 12 European roe deer (Capreolus capreolus). The samples had been collected from legally hunted animals from different districts of Finland during 2008–2009. We analysed the samples for total anti-HEV antibodies using a double-sandwich ELISA assay. Seropositive sera were analysed with RT-qPCR for HEV RNA. HEV seroprevalence was 9.1% (31/342) in moose and 1.4% (1/70) in white-tailed deer. None of the European roe deer were HEV seropositive (0/12). No HEV RNA was detected from samples of seropositive animals. HEV seropositive moose were detected in all districts. Statistically, HEV seroprevalence in moose was significantly higher (p

Ascaris eggs are commonly used as indicators for pathogen inactivation during the treatment of fecal sludge and wastewater due to their highly resistant lipid membrane and ability to survive in the environment for long periods of time. Current guidelines suggest that thermal treatment alone cannot inactivate Ascaris eggs at temperatures below 45 °C, although some evidence in the literature suggests this to be incorrect. Here, we performed a controlled experiment to test the effect of mesophilic temperatures on Ascaris inactivation. We exposed Ascaris suum eggs to a temperature gradient between 34°C and 45 °C under anaerobic and aerobic conditions to observe the required exposure times for a 3-log reduction. Indeed, we found that temperatures lower than 45 °C did inactivate these eggs, and the required exposure times were up to two orders of magnitude shorter than suggested by current guidelines. Results from the anaerobic exposures were used to develop a time-temperature relationship that is appropriate for Ascaris inactivation at mesophilic temperatures. Data from the literature demonstrated that our relationship is conservative, with faster inactivation occurring under environmental conditions when Ascaris eggs were suspended in fecal sludge or manure. A specific aerobic relationship was not developed, but we demonstrated that aerobic conditions cause faster inactivation than anaerobic conditions. Therefore, the anaerobic relationship provides a conservative guideline for both conditions. We demonstrate that relatively low temperatures can considerably impact Ascaris viability and suggest that mesophilic temperatures can be used in waste treatment processes to inactivate pathogens. The development of safe, low-input, mesophilic treatment processes is particularly valuable for ensuring universal access to safe sanitation and excreta management., Graphical abstract Image 1, Highlights • Ascaris eggs were consistently inactivated at mesophilic temperatures of 37–45 °C. • Inactivation is faster and at lower temperatures than current guidelines suggest. • Time-temperature relationship was developed for Ascaris inactivation at 37°C–45 °C. • Thermal inactivation was faster under aerobic conditions. • Results here are conservative compared to inactivation in manure from literature.

SUMMARY The aim of this study is to investigate the bacterial population in coalho goat cheese produced in the semi-arid northeast region of Brazil, to analyse the antibiotic resistance profiles of the identified pathogenic bacteria, to detect the staphylococcal enterotoxin genes and to evaluate the addition of autochthonous lactic acid bacteria (LAB) with technofunctional properties for the control of Staphylococcus aureus growth. In the analysed samples, strains of Escherichia coli (N=11), Salmonella spp. (N=18), Listeria spp. (N=6) and S. aureus (N=9) were classified as multidrug resistant (MDR). The most commonly isolated pathogen from the studied coalho goat cheese was S. aureus. Its isolates were positive for the genes encoding enterotoxins A (sea), B (seb), C (sec) and D (sed). The autochthonous LAB with the potential to inhibit S. aureus were identified as Enterococcus faecium. These strains were selected for in vitro tests of protective, safety, technological and functional properties. In the coalho goat cheese food matrix, these selected autochthonous LAB were able to reduce the enterotoxigenic MDR S. aureus load by approx. 3 log units.

Since the innovation of our new half-piece elastometric respirator, this type of filtering facepiece respirator (FFR) has been used widely in Thailand. Decontamination methods including ultraviolet C (UVC) germicidal irradiation and 70% alcohol have been implemented to decontaminate these respirators. We then examined the inactivation potential of different decontamination processes on porcine epidemic diarrhea virus (PEDV) and numerous bacterial strains, most of which were skin-derived. To enable rigorous integrity of the masks after repeated decontamination processes, fit tests by the Bitrex test, tensile strength and elongation at break were also evaluated. Our results showed that UVC irradiation at a dose of 3 J/cm2 can eradicate bacteria after 60 min and viruses after 10 min. No fungi were found on the mask surface before decontamination. The good fit test results, tensile strength and elongation at break were still maintained after multiple cycles of decontamination. No evidence of physical degradation was found by gross visual inspection. Alcohol (70%) is also an easy and effective way to eradicate microorganisms on respirators. As the current pandemic is expected to continue for months to years, the need to supply adequate reserves of personnel protective equipment (PPE) and develop effective PPE reprocessing methods is crucial. Our studies demonstrated that the novel silicone mask can be safely reprocessed and decontaminated for many cycles by UVC irradiation, which will help ameliorate the shortage of important protective devices in the COVID-19 pandemic era.

The α- and β-class carbonic anhydrases (CAs, EC 4.2.1.1) from the pathogenic bacterium Vibrio cholerae, VchCAα, and VchCAβ, were investigated for their activation with natural and non-natural amino acids and amines. The most effective VchCAα activators were L-tyrosine, histamine, serotonin, and 4-aminoethyl-morpholine, which had KAs in the range of 8.21–12.0 µM. The most effective VchCAβ activators were D-tyrosine, dopamine, serotonin, 2-pyridyl-methylamine, 2-aminoethylpyridine, and 2-aminoethylpiperazine, which had KAs in the submicromolar – low micromolar range (0.18–1.37 µM). The two bacterial enzymes had very different activation profiles with these compounds, between each other, and in comparison to the human isoforms hCA I and II. Some amines were selective activators of VchCAβ, including 2-pyridylmethylamine (KA of 180 nm for VchCAβ, and more than 20 µM for VchCAα and hCA I/II). The activation of CAs from bacteria, such as VchCAα/β has not been considered previously for possible biomedical applications. It would be of interest to study in more detail the extent that CA activators are implicated in the virulence and colonisation of the host by such pathogenic bacteria, which for Vibrio cholerae, is highly dependent on the bicarbonate concentration and pH in the surrounding tissue.

Bats are unique mammals that are reservoirs of high levels of virus diversity. Although several of these viruses are zoonotic, the majority are not. Astroviruses, transmitted fecal-orally, are commonly detected in a wide diversity of bat species, are prevalent at high rates and are not thought to directly infect humans. These features make astroviruses useful in examining virus evolutionary history, epidemiology in the host, and temporal shedding trends. Our study screened for the presence of astroviruses in bats in Singapore, reconstructed the phylogenetic relations of the polymerase genes and tested for population characteristics associated with infection. Of the seven species screened, astroviruses were detected in Rhinolophus lepidus and Eonycteris spelaea. The R. lepidus sequences grouped with other Rhinolophus astrovirus sequences from China and Laos, while the Eoncyteris sequences formed a distinct clade with astroviruses from Rousettus spp. in Laos and Pteropus giganteus in Bangladesh, but not with other E. spelaea sequences. Longitudinal collections of Eonycteris feces demonstrated variable shedding. Juvenile status of bats was a risk factor for astroviruses. This study highlights the diversity of astroviruses in nectivorous and insectivorous bats in Singapore and provides a predictive framework for understanding astrovirus infection in these bats. It also suggests that in addition to host phylogenetic relatedness, host ecology, such as roosting behavior, may drive co-infections, virus maintenance and spillover.

SUMMARYSeasonal respiratory illnesses present a major burden on primary care services. We assessed the burden of respiratory illness on a national telehealth system in England and investigated the potential for providing early warning of respiratory infection. We compared weekly laboratory reports for respiratory pathogens with telehealth calls (NHS 111) between week 40 in 2013 and week 29 in 2015. Multiple linear regression was used to identify which pathogens had a significant association with respiratory calls. Children aged

Congenital cytomegalovirus (CMV) infection is a major cause of birth defects ranging from developmental disorders to stillbirth. Most newborns affected by CMV do not present with symptoms at birth but are at risk of sequelae at later stages of their childhood. Stored dried blood spots (DBS) taken at birth can be used for retrospective diagnosis of hereditary diseases, but detection of pathogens is challenged by potentially low pathogen concentrations in the small blood volume available in a DBS. Here we test four different extraction methods for optimal recovery of CMV DNA from DBS at low to high CMV titers. The recovery efficiencies varied widely between the different extractions (from 3% to 100%) with the most efficient method extracting up to 113-fold more CMV DNA than the least efficient and 8-fold more than the reference protocol. Furthermore, we amplified four immunomodulatory CMV genes from the extracted DNA: the UL40 and UL111A genes which occur as functional knockouts in some circulating CMV strains, and the highly variable UL146 and US28 genes. The PCRs specifically amplified the CMV genes at all tested titers with sufficient quality for sequencing and genotyping. In summary, we here report an extraction method for optimal recovery of CMV DNA from DBSs that can be used for both detection of CMV and for genotyping of polymorphic CMV genes in congenital CMV infection.

Recycling post-hydrothermal liquefaction wastewater (PHWW) may allow the use of nutrients in the aqueous phase that may otherwise go unused. PHWW is an attractive option for use as fertilizer in systems like crop production. However, there are potential contaminants in the PHWW that may inhibit crop growth or pose a food safety risk. This study investigated the concentrations of heavy metals and nutrients in the PHWW, as well as the presence of indicator pathogens. In addition, four different water treatment methods were used: (1) dilution of raw PHWW, (2) sand filtration after dilution, (3) sand and carbon filtration after dilution, and (4) reverse osmosis after dilution. Our results indicate that the concentrations of cadmium, lead, and arsenic in raw PHWW were well below the maximum recommended concentrations set by the US Environmental Protection Agency (US EPA) for Water Reuse. In addition, the treatment methods in this study achieved percent removals ranging from 82&ndash, 100% for cadmium, 99&ndash, 100% for mercury, 75&ndash, 99.5% for lead, and 71&ndash, 99% for arsenic. Nitrogen in raw PHWW was predominantly in the total N form, preventing it from being accessible to plants. After nitrification was induced, the concentration of NO3 + NO2 increased by 1.75 mg/L in the untreated 5% PHWW mixture, but remained unchanged or decreased for all other treatments and mixtures. There were no E. coli or coliform colonies detected in the raw PHWW, or in any PHWW mixtures. All PHWW mixtures with and without treatment are within US EPA guidelines for metals for irrigation water reuse. However, fertilizer supplementation may be required for PHWW to be suitable for crop production, as the low concentrations of NO3 + NO2 may prove challenging for growing crops.

An electrochemically activated solution (ECAS), generated by electrolysis of a dilute sodium chloride solution in a four-chamber electrolytic cell (Ecas4), was tested as a sanitising aerosol in eliminating bacteria from the environment of a weaning room vacated 24-48h earlier, at a continuous flow pig farm. An ultrasonic humidifier was used to fill the environment with a fog (droplets with diameters of 1–5 μm) containing 0.25 ppm of hypochlorous acid. The weaning room was fogged for 3 min at 30 min intervals during five hours of aerosol disinfection. An innovative sample treatment with propidium monoazide dye in conjunction with cyclonic air sampling was optimised and adapted for discerning live/dead bacteria in subsequent molecular quantification steps. Without fogging, total bacterial load ranged from 5.06 ± 0.04 to 5.75 ± 0.04 Log10 CFU/m3. After the first hour of fogging, a 78% total bacterial reduction was observed, which further increased to > 97% after the second hour, > 99.4% after the third and 99.8% after the fourth hour, finally resulting in a 99.99% reduction from the farm environment over five hours. Unlike the current formaldehyde spray disinfection protocol, which requires a long empty period because of its hazardous properties, this economically viable and environmentally friendly disinfection protocol may significantly lower downtime. Moreover, ECAS fogging can be easily adapted to a variety of applications, including the elimination of pathogens from livestock farm air environment for disease prevention, as well as decontamination after disease outbreaks. Refereed/Peer-reviewed

The response to the COVID-19 epidemic is generating severe shortages of personal protective equipment around the world. In particular, the supply of N95 respirator masks has become severely depleted, with supplies having to be rationed and health care workers having to use masks for prolonged periods in many countries. We sought to test the ability of 7 different decontamination methods: autoclave treatment, ethylene oxide gassing (ETO), low temperature hydrogen peroxide gas plasma (LT-HPGP) treatment, vaporous hydrogen peroxide (VHP) exposure, peracetic acid dry fogging (PAF), ultraviolet C irradiation (UVCI) and moist heat (MH) treatment to decontaminate a variety of different N95 masks following experimental contamination with SARS-CoV-2 or vesicular stomatitis virus as a surrogate. In addition, we sought to determine whether masks would tolerate repeated cycles of decontamination while maintaining structural and functional integrity. All methods except for UVCI were effective in total elimination of viable virus from treated masks. We found that all respirator masks tolerated at least one cycle of all treatment modalities without structural or functional deterioration as assessed by fit testing; filtration efficiency testing results were mostly similar except that a single cycle of LT-HPGP was associated with failures in 3 of 6 masks assessed. VHP, PAF, UVCI, and MH were associated with preserved mask integrity to a minimum of 10 cycles by both fit and filtration testing. A similar result was shown with ethylene oxide gassing to the maximum 3 cycles tested. Pleated, layered non-woven fabric N95 masks retained integrity in fit testing for at least 10 cycles of autoclaving but the molded N95 masks failed after 1 cycle; filtration testing however was intact to 5 cycles for all masks. The successful application of autoclaving for layered, pleated masks may be of particular use to institutions globally due to the virtually universal accessibility of autoclaves in health care settings. Given the ability to modify widely available heating cabinets on hospital wards in well-resourced settings, the application of moist heat may allow local processing of N95 masks.

As in the traditional production of cheese in lambskin sacks raw cow’s or sheep’s milk is mostly used, the purpose of this study is to see how the production affects the microbiological quality of the cheese. To do that, we tested 39 samples of raw cow’s and sheep’s milk, curd, ripened cheese (15, 30 and 45 days) and lambskin sacks for native microbial population. Two-thirds of the milk, curd and cheese samples had higher counts of staphylococci and enterobacteria than permitted by regulations. Not a single sample had Salmonella and Listeria monocytogenes, but we did find Escherichia coli in sheep’s milk and cheese, and yeast and mould in both types of milk and cheese. Staphylococcus xylosus prevailed in lambskin sacks. Despite the high incidence of S. aureus, even in the final product, staphylococcal enterotoxin was detected in only two sheep’s cheese samples. Among the lactic acid bacteria, Lactococcus lactis and Lactobacillus paracasei prevailed in cow’s cheese, whereas Leuconostoc mesenteroides and Lactobacillus plantarum prevailed in sheep’s cheese. In the lambskin sacks Leuconostoc mesenteroides and Lactobacillus plantarum were predominant. Our findings give an important insight into the fermentation and microbial ecology of the cheese in lambskin sacks.

A review of the literature published in 2013 on topics relating to the fate of emerging environmental pollutants during wastewater treatment is presented. This review is divided into the following sections: emerging biological agents, estrogens, metals, nanomaterials, nutrients, persistent organic pollutants, pharmaceuticals and personal care products and herbicide, and trace organic contaminants.

Supertypes are groups of human leukocyte antigen (HLA) alleles which bind overlapping sets of peptides due to sharing specific residues at the anchor positions—the B and F pockets—of the peptide-binding region (PBR). HLA alleles within the same supertype are expected to be functionally similar, while those from different supertypes are expected to be functionally distinct, presenting different sets of peptides. In this study, we applied the supertype classification to the HLA-A and HLA-B data of 55 worldwide populations in order to investigate the effect of natural selection on supertype rather than allelic variation at these loci. We compared the nucleotide diversity of the B and F pockets with that of the other PBR regions through a resampling procedure and compared the patterns of within-population heterozygosity (He) and between-population differentiation (GST) observed when using the supertype definition to those estimated when using randomized groups of alleles. At HLA-A, low levels of variation are observed at B and F pockets and randomized He and GST do not differ from the observed data. By contrast, HLA-B concentrates most of the differences between supertypes, the B pocket showing a particularly high level of variation. Moreover, at HLA-B, the reassignment of alleles into random groups does not reproduce the patterns of population differentiation observed with supertypes. We thus conclude that differently from HLA-A, for which supertype and allelic variation show similar patterns of nucleotide diversity within and between populations, HLA-B has likely evolved through specific adaptations of its B pocket to local pathogens. Electronic supplementary material The online version of this article (doi:10.1007/s00251-015-0875-9) contains supplementary material, which is available to authorized users.

In this Research Communication we investigate the microbiological profile of 12 dairy wastewater streams from three contrasting Irish dairy processing factories to determine whether faecal indicators/pathogens were present and in turn, whether disinfection may be required for potential water reuse within the factory. Subsequently, the impact of suspended solids on the inactivation efficiency of Escherichia coli via two means of ultravoilet (UV) disinfection; flow-through pulsed UV (PUV) and continuous low pressure UV (LPUV) disinfection was analysed. Faecal indicators total coliforms and E. coli were detected in 10 out of the 12 samples collected at the dairy processing factories while pathogenic bacteria Listeria monocytogenes was detected in all samples collected at 2 out of the 3 factories. Salmonella spp. was undetected in all samples. The results also indicated that organic dairy wastewater solids had an impact on the performance efficiency of the PUV system and, to a lesser extent, the LPUV system. The findings indicate that the targeting of key pathogens would be required to enable wastewater reuse (and indeed effluent discharges if regulation continues to become more stringent) and that LPUV may offer a more robust disinfection method as it appears to be less susceptible to the presence of suspended solids. The authors would like to acknowledge the Department of Agriculture, Food and the Marine for the funding of this research (Ref: 13-F-507). Additionally, the DairyWater research team would like to the members of the Irish dairy sector for their cooperation and contributions throughout the research project peer-reviewed